Methods for the selection of binding proteins

ABSTRACT

This application provides an improved screening method for the selection of target-binding proteins having desirable biophysical properties. The method combines mRNA display and yeast surface display in a way that takes advantage of the desirable attributes of both processes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of International Application No.PCT/US2012/038032 filed May 16, 2012, which claims priority to U.S.Provisional application No. 61/487,032 filed May 17, 2011The entirecontents of the referenced applications are herein incorporated byreference in their entirety.

BACKGROUND

The production of polypeptides with target protein-binding properties isof great economic interest. At present, a number of methods have beendeveloped to screen large libraries and identify polypeptides withbinding affinity to a protein of interest. Typically, a large number ofgenetically diverse polypeptide mutants are produced in a first step. Ina second step these polypeptide mutants are selected according to theirbinding affinity to the protein of interest. This process for theproduction of diversity and subsequent targeted selection can berepeated as often as it is desired.

However, a protein that has high target binding affinity may not havesufficiently high stability and solubility. Even for those proteins thathave high solubility, high concentration liquid formulations tend tohave short shelf lives and may lose biological activity as a result ofchemical and physical instabilities during the storage. For example,aggregate formation during storage of a liquid protein composition canadversely affect the biological activity of the protein, resulting inloss of therapeutic efficacy and/or an increase in immunogenicity inhumans.

This application provides an improved screening method for the selectionof target-binding proteins having desirable biophysical properties(e.g., high solubility, stability, etc.). The method combines mRNAdisplay and yeast surface display in a way that takes advantage of thedesirable attributes of both processes.

SUMMARY

Provided herein are methods for selecting at least one target-bindingprotein with favorable biophysical properties. A method may comprise (i)a first phase comprising selecting a first population of proteinvariants binding a target protein (“target-binding protein variants”)from a library comprising at least about 10¹² protein variants; and (ii)a second phase comprising selecting a second population oftarget-binding protein variants from at least a portion of the firstpopulation of target-binding protein variants using yeast surfacedisplay, wherein the second population of target-binding proteinvariants comprises at least one target-binding protein with favorablebiophysical properties. The library may comprise at least about 10¹³protein variants. The first phase may comprise at least one round ofselection, wherein a round of selection comprises contacting the targetprotein with the library of protein variants, removing any proteinvariants that do not bind to the target with a desired affinity, andisolating the remaining protein variants. The first phase may compriseat least 2 consecutive rounds of selection. The first phase may compriseat least 3 to 4 consecutive rounds of selection. The first phase maycomprise a number of rounds of selection sufficient to obtain at most10⁴ to 10⁸ target-binding protein variants, e.g., 10⁵ to 10⁷target-binding protein variants. The library of the first phase may be alibrary of mRNA-protein fusions. The mRNA-protein fusions may becovalently conjugated via a puromycin linker. The second phase maycomprise at least one round of selection, wherein a round of selectioncomprises contacting the target protein with the first population oftarget-binding protein variants displayed on yeast cells, removing anyyeast cells displaying protein variants that do not bind to the targetwith a desired affinity, to thereby select the second population oftarget-binding protein variants. The second phase may comprise at least2 consecutive rounds of selection. The second phase may comprise atleast 3 to 6 consecutive rounds of selection. The second phase maycomprise a number of rounds of selection sufficient to obtain at most10³ (or at most 10²) target-binding protein variants. The at least aportion of the first population of target-binding protein variants inthe second phase may comprise at least 10⁵ target-binding proteinvariants. In an exemplary embodiment, the first phase comprisesproviding a library comprising at least 10¹² protein variants andperforming a number of rounds of selection of the library sufficient toobtain from 10⁴ to 10⁸ (e.g., 10⁵ to 10⁷) target-binding proteinvariants; and the second phase comprises performing a number of roundsof selection on at least a portion of the 10⁴ to 10⁸ (e.g., 10⁵ to 10⁷)target-binding protein variants of the first phase sufficient to obtainat most about 10³ target-binding protein variants. The second phase maycomprise performing a number of rounds of selection sufficient to obtainat most about 10² target-binding protein variants. The second phase maycomprise at least one round of selection wherein at most the top 10% ofthe yeast cells that express the highest level of target-binding proteinvariants are selected. Each of the rounds of selection of yeast surfacedisplay may comprise selecting at most the top 10% of the yeast cellsthat express the highest level of target-binding protein variants. Amethod may further comprise affinity maturation during or after thefirst phase, during or after the second phase, or both. The favorablebiophysical properties may be one or more of solubility,thermostability, high expression levels, low levels of aggregation,proper folding, or binding specificity. The method may increase thelikelihood of selecting a target-binding protein variant that ismonomeric by at least two-fold relative to a method of selection thatdoes not comprise a second phase using yeast display. The method mayincrease the likelihood of selecting a target-binding protein varianthaving a higher melting temperature relative to a method of selectionthat does not comprise a second phase using yeast display. In certainembodiments, the library of protein variants is a library of ¹⁰Fn3variants. The ¹⁰Fn3 variants may comprise sequence modifications in theBC, DE or FG loops. The variants may comprise sequence modifications inat least one loop and at least one beta strand. One or more of the BC,DE and FG loops may be extended in length or reduced in length relativethe length of the corresponding loop of the wild-type human ¹⁰Fn3domain. In a particular example, the first phase comprises providing alibrary comprising at least 10¹² protein variants and performing anumber of rounds of selection of the library sufficient to obtain from10⁴ to 10⁸ (e.g., 10⁵ to 10⁷) target-binding protein variants; and thesecond phase comprises performing a number of rounds of selection on atleast a portion of the 10⁴ to 10⁸ (e.g., 10⁵ to 10⁷) target-bindingprotein variants sufficient to obtain at most about 10³ (e.g., at most10²) target-binding protein variants, and wherein the library of proteinvariants is a library of ¹⁰Fn3 variants, wherein each protein variantcomprises at least one modification in a loop and at least onemodification in a beta strand relative to a wild-type ¹⁰Fn3 protein. Thelibrary of the first phase is a mRNA-display library.

Further provided herein are methods for selecting a target-bindingprotein with favorable biophysical properties comprising (i) providingfrom 10⁴ to 10⁸ (e.g., 10⁵ to 10⁷) target-binding protein variantsselected from a library of protein variants comprising at least 10¹²members; and (ii) selecting a population of at most 10³ (e.g., at most10²) target-binding protein variants from at least10⁵ target-bindingprotein variants of step (i) using yeast surface display. The 10⁴ to 10⁸(e.g., 10⁵ to 10⁷) target-binding protein variants of step (i) may beselected from a library of protein variants comprising at least 10¹³members.

Also encompassed herein are methods for selecting a target-bindingprotein with favorable biophysical properties comprising (i) selecting afirst population of target-binding protein variants consisting of atmost 10³ (e.g., at most 10²) target-binding protein variants from alibrary comprising at least about 10¹² protein variants, comprisingconducting a number of rounds of selection sufficient to obtain the atmost 10³ (e.g., at most 10²) target-binding protein variants; (ii)determining whether a significant number of the at most 10³ (e.g., atmost 10²) target-binding protein variants have favorable biophysicalproperties; and (iii) if a significant number of the at most 10³ (e.g.,at most 10²) target-binding protein variants do not have favorablebiophysical properties, then selecting a population of at most 10³(e.g., at most 10²) target-binding protein variants from at least 10⁵target-binding protein variants obtained during the selection in step(i) using yeast surface display. “Significant number” may refer to atleast about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%. Selecting apopulation of at least 10³ (e.g., at most 10²) target-binding proteinvariants from at least 10⁵ target-binding protein variants obtainedduring the selection in step (i) using yeast surface display maycomprise selecting at most the top 10% of the yeast cells that expressthe highest level of target-binding protein variants. Each of the roundsof selection of yeast surface display may comprise selecting at most thetop 10% of the yeast cells that express the highest level oftarget-binding protein variants.

The application provides a two-phase method for selecting one or moretarget-binding protein variants with favorable biophysical properties.Any type of protein may be screened according to the methods describedherein, including, for example, scFvs, Fabs or ¹⁰Fn3 domain proteins. Inan exemplary embodiment, the method involves screening a library of¹⁰Fn3 domain protein variants. The library may comprise a plurality of¹⁰Fn3 domain proteins having sequence modifications in one or more ofthe BC, DE and/or FG loops. In certain embodiments, one of more of theBC, DE and/or FG loops may be extended in length or reduced in lengthrelative to the corresponding loop of the wild-type human ¹⁰Fn3 domain.

In certain embodiments, the first phase involves screening a library ofRNA-protein fusions, wherein the mRNA-protein fusions are covalentlyconjugated via a puromycin linker.

In one aspect, the two-phase method comprises screening a library ofprotein variants against a target protein using mRNA display to select apopulation of target-binding protein variants. The second phasecomprises screening the population of target-binding protein variantsagainst the target protein using yeast surface display to select asub-population of target-binding protein variants with favorablebiophysical properties.

In certain embodiments, first phase comprises screening a librarycomprising at least 10¹² protein variants using mRNA display.

In certain embodiments, the first phase of screening involves contactingthe target protein with the library of protein variants, removing anyprotein variants that do not bind to the target with a desired affinity,and isolating the remaining protein variants.

In certain embodiments, the first phase of screening comprises the stepsof a) producing a population of candidate RNA molecules that encode aplurality of candidate protein variant sequences, each RNA comprising atranslation initiation sequence and a start codon operably linked tosaid candidate protein variant coding sequence and each being operablylinked to a peptidyl acceptor at the 3′ end; b) in vitro translatingsaid candidate protein variant coding sequences to produce a populationof candidate RNA-protein fusions; c) contacting said population ofcandidate RNA-protein fusions with a target protein; d) selecting apopulation of target-binding RNA-protein fusions, the protein portion ofwhich has a binding affinity or specificity for the target protein; ande) recovering the genetic information encoding the population oftarget-binding protein variants by the reverse transcribing the RNAportion to produce complementary DNA.

In certain embodiments, the first phase of screening is repeated atleast 2 consecutive times. In certain embodiments, the first phase ofscreening is repeated at least 3 to 4 consecutive times.

In certain embodiments, the second phase of screening involvescontacting the target protein with the population of protein variantsselected in the first phase, removing any protein variants that do notbind to the target with a desired affinity, and isolating the remainingprotein variants.

In certain embodiments, the second phase of screening comprises thesteps of a) displaying the population of protein variants on the surfaceof yeast cells, b) contacting the yeast cells with a target protein; andc) sorting the yeast cells based on the binding of the target proteinthereby isolating the yeast cells expressing protein variants having thedesired binding affinity for the target.

In certain embodiments, the second phase of screening is repeated atleast 2 consecutive times. In certain embodiments, the second phase ofscreening is repeated at least 3 to 6 consecutive times.

In certain embodiments, the sub-population of target-binding proteinvariants with favorable biophysical properties comprises at least 100protein variants.

In certain embodiments, the method further comprises affinity maturationfollowing the first phase of screening, following the second phase ofscreening, or both. In certain embodiments, the method further comprisesaffinity maturation during the first phase of screening, during thesecond phase of screening, or both.

In certain embodiments, the favorable biophysical properties are one ormore of solubility, thermostability, high expression levels, low levelsof aggregation, proper folding and/or binding specificity.

In certain embodiments, the method increases the likelihood of selectinga target-binding protein variant that is monomeric by at least two-foldrelative to a method involving screening the same library using onlymRNA display.

In certain embodiments, the method increases the likelihood of selectinga target-binding protein variant having a higher melting temperaturerelative to a method involving screening the same library using onlymRNA display.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1. Yeast population labeled in round 1 of yeast surface display.Relative “Display level” is revealed by fluorescent labeling of theC-terminal epitope tag, and “Binding” is quantified by labeling withfluorescently labeled PCSK9. The gate labeled “P2” contains yeast cellsthat are decorated with ¹⁰Fn3 domain proteins that both display at ahigh level and bind PCSK9; the cells in this gate were captured byfluorescence-activated cell sorting and used as the starting point ofround 2 of yeast-display sorting.

FIG. 2. Comparison of state of aggregation between ¹⁰Fn3 domain proteinpopulations selected for binding to PCSK9 by mRNA display only (“R”;left bar of each double bar) versus by combination of mRNA display andyeast surface display (“R+Y”; right bar of each double bar). Theproteins scored as “4” are predominantly aggregated, the proteins scoredas “3” are a mixture of oligomer and monomer, and the proteins scored as“2” are mostly monomeric, with a minor fraction of oligomer. None of theproteins tested were completely monomeric.

FIG. 3. Comparison of thermostability between ¹⁰Fn3 domain proteinpopulations selected for binding to PCSK9 by mRNA display only (“R”;left bar of each double bar) versus by combination of mRNA display andyeast surface display (“Y”; right bar of each double bar). Meltingtemperatures (“T_(m)”) were determined by differential scanningcalorimetry.

FIG. 4. Yeast population labeled in round 3 of yeast surface display.Relative “Display level” is revealed by fluorescent labeling of theC-terminal epitope tag, and “Binding” is quantified by labeling withfluorescently labeled target. The gate labeled “P3” contains yeast cellsthat are decorated with ¹⁰Fn3 domain proteins that both display at ahigh level and bind the target; the cells in this gate were captured byfluorescence-activated cell sorting and used as the starting point ofround 4 of yeast-display sorting.

FIG. 5. Comparison of state of aggregation between ¹⁰Fn3 domain proteinpopulations selected for binding to the target by mRNA display only(“R”; left bar of each double bar) versus by combination of mRNA displayand yeast surface display (“R+Y”; right bar of each double bar). Theproteins scored as “4” are predominantly aggregated, the proteins scoredas “3” are a mixture of oligomer and monomer, the proteins scored as “2”are mostly monomeric, with a minor fraction of oligomer, and proteinsscored as “1” are completely monomeric.

FIG. 6. Comparison of thermostability and state of aggregations between¹⁰Fn3 domain protein populations selected for binding to the target bymRNA display only (“R”) versus by combination of mRNA display and yeastsurface display (“Y”). Half-points of thermal transition (“T_(H)”) weremonitored by change in fluorescence upon heating.

DETAILED DESCRIPTION

Overview

This application relates to an improved screening method for theselection of target-binding proteins having desirable biophysicalproperties. The method combines mRNA display and yeast surface displayin a way that takes advantage of the desirable attributes of bothprocesses. First, mRNA display technology is used to screen a largelibrary of nucleic acid-protein fusions having high design complexity.Typically, the mRNA display phase of the selection method lasts for twoto ten rounds and focuses on the removal of variants that do not bindthe target of interest with desirable affinity. Once the population ofvariants has been reduced to a desired level of complexity, it istransferred to a yeast surface display system, which proceeds foradditional rounds of screening. The yeast surface display phase of theselection method is designed to introduce selection stringency for ahigh display level, and thus selects target-binding candidates with highstability, high protein expression, proper folding, and low levels ofaggregation. Accordingly, the screening methods disclosed herein areadvantageous, in part, because (i) the mRNA display phase permits thescreening of large libraries, thus increasing the probability ofcapturing target-binding protein variants having the desired targetbinding affinity, and (ii) the yeast surface display phase removes thevariants with poor biophysical properties. Thus the odds of selecting avariant protein having the desired biophysical and binding propertiesare higher when using the combination method disclosed herein than whenusing mRNA display technology or yeast display on its own.

The methods described herein may be used to isolate any type of bindingprotein that is compatible with mRNA display and yeast display. Incertain embodiments, the binding proteins are small, single chainpolypeptides. Exemplary binding proteins that can be selected using themethods described herein include, for example, scFv proteins, Fabproteins and ¹⁰Fn3 domain proteins.

The methods described herein may be used to isolate a binding proteinthat binds to any target of interest. Exemplary targets are therapeuticproteins, such as, for example, PCSK9, VEGFR2, IGF-1R, TNFα and EGFR.Proteins binding to such targets may be used in place of antibodytherapeutics that bind to the same target. For example, proteins thatbind to TNFα may be used to treat autoimmune disorders such asrheumatoid arthritis, inflammatory bowel disease, psoriasis, and asthma;proteins that bind to VEGFR2, IGF-1R, and EGFR may be used to treathyperproliferative disorders or diseases associated with unwantedangiogenesis, such as cancers or tumors; and proteins that bind to PCSK9may be used to treat atherosclerosis, hypercholesterolemia and othercholesterol related diseases. Alternatively, binding proteins selectedusing the methods described herein may be used to detect the targetprotein (either in vitro or in vivo, e.g., when fused to a detectablelabel) or to purify the target protein (e.g., by isolating the bindingon a solid support such as a column resin).

In exemplary embodiments, a library is first screened using mRNAdisplay. The library used for the mRNA display may contain at least10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴ or 10¹⁵ members, or from 10¹⁰ to 10¹⁵, 10¹⁰to 10¹⁴, 10¹⁰ to 10¹², 10¹¹to 10¹³, 10¹² to 10¹⁴ or from 10¹² to 10¹³members. In certain embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9 or 10rounds of mRNA display screening is carried out, or from 2-10, 2-8, 2-7,2-6, 2-5, 2-4, 2-3, 3-10, 3-8, 3-6, 3-5, 3-4, 4-10, 4-8, or 4-6 roundsof mRNA display screening is conducted. In certain embodiments,iterative rounds of mRNA display screening are carried out until thediversity of the library is simplified to less than about 10¹⁰, 10⁹,10⁸, 10⁷, 10⁶, 10⁵, 10⁴ members, or from 10⁵-10¹⁰, 10⁵-10⁸, 10⁵-10⁷,10⁵-10⁶ members. A round of mRNA display screening refers to contactingan immobilized target with the library, washing the mixture to removeunbound library members, and isolating the bound mRNA-protein fusionlibrary members. The result of the mRNA display screening is a lessdiverse library that has been enriched for protein variants that bind tothe desired target.

In exemplary embodiments, the enriched library of clones from mRNAdisplay is then subjected to additional rounds of screening using ayeast surface display technique. The enriched library of clones maycontain less than about 10¹⁰, 10⁹, 10⁸, 10⁷, 10⁶, 10⁵, 10⁴ members, orfrom 10⁵-10¹⁰, 10⁵-10⁸, 10⁵-10⁷, 10⁵-10⁶ members. In certainembodiments, at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 rounds of yeastdisplay screening are carried out, or from 2-10, 2-8, 2-7, 2-6, 2-5,2-4, 2-3, 3-10, 3-8, 3-6, 3-5, 3-4, 4-10, 4-8, or 4-6 rounds of yeastdisplay screening are conducted. In certain embodiments, iterativerounds of yeast display screening are carried out until the diversity ofthe library is simplified to less than about 10 ⁴, 10³, or 10² members,or from 10²-10⁴ or 10²-10³ members. In certain embodiments, iterativerounds of yeast display are carried out until one or more proteinvariants having desired binding and biophysical characteristics areidentified from the library. A round of yeast display screening refersto contacting yeast decorated with target binding proteins with alabeled target and selecting those yeast displaying protein variantsthat have a desired level of binding to the target. The result of theyeast display screening is the identification of one or more clones thatbind to the target with a desired level of affinity and that havedesirable biophysical properties such as thermostability, properfolding, little to no aggregation, and high levels of expression.

In certain embodiments, the library may be subject to affinitymaturation at one or more times during the method. For example, geneshuffling and/or error-prone PCR may be applied during the mRNA displayscreening, the yeast display screening, or during both or between thetwo screening procedures in order to introduce additional diversity.

In certain embodiments, the methods described herein increase thelikelihood of selecting a target binding protein having one or moredesirable biophysical or biochemical properties, such as, for example,one or more of solubility, thermostability, high expression levels, lowlevels of aggregation, proper folding and/or binding specificity. Incertain embodiments, the methods described herein increase thelikelihood of selecting a target-binding protein variant that ismonomeric by at least two-fold, three-fold, four-fold, five-fold,ten-fold or more, or from 2 to 5 fold, or from 2 to 3 fold relative to amethod involving screening the same library using only mRNA display. Incertain embodiments, the methods described herein result in theselection of a population of target binding protein variants having anaverage melting temperature (Tm) that is higher than the average meltingtemperature of the population of target binding protein variantsselected by screening the same library using only mRNA display.

mRNA Display

mRNA display is a mRNA-protein fusion technology that covalently couplesa protein to its encoding genetic material. mRNA display technology maybe used to generate large libraries that can contain as many as 10¹³different variants. For a more detailed description of mRNA displaytechnology see Szostak et al., U.S. Pat. Nos. 6,258,558, 6,261,804,6,214,553, 6,281,344, 6,207,446, 6,518,018; PCT Publication Nos. WO00/34784, WO 01/64942, WO 02/032925; and Roberts et al., Proc Natl.Acad. Sci., 94:12297-12302 (1997), herein incorporated by reference.

Because an mRNA-display library is constructed exclusively in vitro, itcan contain as many as 10¹³ different variants, two to five orders ofmagnitude higher than what is accessible to methods that require thetransformation of a host microorganism. Suitable examples of cell-freeprotein synthesis systems that may be used in connection with mRNAdisplay include, for example, wheat germ extract, rabbit reticulocytelysate, or Escherichia coli S30 extract. Since ¹⁰Fn3 contains fewer than100 amino-acid residues, the DNA segment encoding the library of ¹⁰Fn3variants can be easily assembled by PCR from eight to twelve overlappedoligonucleotides, some of which contain randomized or partiallydiversified sequences. It may be advantageous to design thelibrary-construction strategy so that the length of diversifiedoligonucleotides is minimized, to use the highest qualityoligonucleotides available, and to assemble them using a high-fidelitypolymerase. The amount of PCR primers and the number of cycles duringthe amplification of diversified library fragments and full-lengthlibrary should be limited to obtain the desired number of copies of eachclone, while limiting the number of unplanned mutations.

Once the library is assembled, it is then transcribed into mRNA using anin vitro transcription system. The mRNA is then chemically linked to aDNA oligonucleotide that contains a peptidyl adaptor molecule, such asthe tRNA-like molecule puromycin, and subjected to an in vitrotranslation system. After the mRNA is translated into a polypeptide, thepeptidyl acceptor molecule conjugated to the mRNA inserts into theribosome and is added to the nascent polypeptide chain, as if it werethe last amino-acid residue in the chain. The resulting covalentmRNA-protein fusion may be reverse transcribed to add a stabilizing,complimentary DNA strand. The mRNA-protein fusion molecules are purifiedaway from the in vitro translation reaction mixture using anyart-recognized methods of separation of mRNA-protein fusion moleculesfrom a reaction mixture. In some embodiments, the mRNA-protein fusionsare separated by chromatography using a polydeoxythimidine (polydT)resin. In certain embodiments, the mRNA-protein fusions are separated bybinding to an antibody specific for an epitope present in the proteincomponent of the mRNA-protein fusion. The epitope may be an amino acidsequence tag, for example, FLAG® or HA tags, incorporated into the aminoacid sequence of the protein component of the mRNA-protein fusion, forexample, at the N-terminal or C-terminal region.

A peptidyl acceptor refers to any molecule capable of being added to theC-terminus of a growing protein chain by the catalytic activity of theribosomal peptidyl transferase function. Typically, such moleculescontain (i) a nucleotide or nucleotide-like moiety (for example,puromycin and analogues thereof), (ii) an amino acid or amino acid-likemoiety (for example, any of the 20 D- or L-amino acids or any amino acidanalog thereof (for example, 0-methyl tyrosine or any of the analogsdescribed by Ellman et al., Meth. Enzymol., 202:301 (1991)), and (iii) alinkage between the two (for example, an ester, amide, or ketone linkageat the 3′ position or, less preferably, the 2′ position); preferably,this linkage does not significantly perturb the structure of the ringfrom the natural ribonucleotide conformation. In addition, this termencompasses, without limitation, a peptide acceptor molecule that iscovalently bonded (either directly or indirectly through interveningnucleic acid sequence) to the protein coding sequence, as well as onethat is joined to the protein coding sequence by some non-covalentmeans, for example, through hybridization using a second nucleic acidsequence that binds at or near the 3′ end of the protein coding sequenceand that itself is bound to a peptide acceptor molecule.

In some embodiments, the peptidyl acceptor is a tRNA-like structureother than puromycin. Such compounds include, without limitation, anycompound which possesses an amino acid linked to an adenine or anadenine-like compound, such as the amino acid nucleotides,phenylalanyl-adenosine (A-Phe), tyrosyl adenosine (A-Tyr), and alanyladenosine (A-Ala), as well as amide-linked structures, such asphenylalanyl 3′ deoxy 3′ amino adenosine, alanyl 3′ deoxy 3′ aminoadenosine, and tyrosyl 3′ deoxy 3′ amino adenosine; in any of thesecompounds, any of the naturally-occurring L-amino acids or their analogsmay be utilized. In addition, a combined tRNA-like 3′structure-puromycin conjugate may also be used in the invention.

The library is then contacted with a target of interest. The selectionprocess is carried out in an aqueous medium wherein parameters such astime, temperature, pH, buffer, salt concentration, and detergentconcentration may be varied according the stringency of the selectionstrategy employed.

To enrich an mRNA-displayed library of protein variants for those thatbind the target of interest, the library is incubated with the target.Typically, the target molecule is captured on a solid surface such as abead. In some embodiments, a capturable moiety is present on the targetmolecule and is used to immobilize the target on bead. The capturablemoiety may be biotin, an Fc domain, a purification tag, or any othercapturable moiety known to those skilled in the art. The beads may becoated with streptavidin, protein A, or tag-specific antibody, or anyother agent that will capture the capturable-moiety. After themRNA-displayed library is incubated with the bead-captured target, themRNA-displayed molecules that bind to the target remain associated withthe bead-captured target, and the non-binding molecules are removed asflow-through and wash. Alternatively, the target can be conjugateddirectly to a solid support. Exemplary solid supports include, forexample, an epoxy resin, an agarose column, a SEPHAROSE® column, or aBIACORE® chip. The mRNA-display library is then mixed with theimmobilized target, and the reaction is washed, leaving the target-boundvariants bound to the immobilized target. In either case, concentratedbase is added to degrade the mRNA, thereby releasing the DNA encodingthe proteins variants that bound to the target. High-fidelity PCR isused to amplify the captured DNA, now enriched for sequences encodingtarget-binding variants. The enriched population is ready to betranscribed, conjugated to an acceptor molecule, translated, andintroduced into the next round of selection. The mRNA-display selectioncycle is typically repeated for at least 4 to 6 rounds in order toselect/enrich those fusion molecules comprising polypeptide sequencesexhibiting affinity for the target material.

Once an enriched population of mRNA-displayed protein variants showsdetectable binding to target under conditions of desired stringency, theencoding DNAs may be purified and subsequently PCR-amplified using ahigh fidelity polymerase. The DNA fragments may be sequenced and thevariants of interest may be cloned into an expression vector, expressedin a suitable expression system, for example, a bacterial, yeast, insector mammalian system, purified as protein, and characterized. At thispoint, the DNA encoding the variants that bind the target with highaffinity may also be transferred to the yeast surface display systemdetailed below.

In some embodiments, measures are taken to avoid selecting proteinvariants that bind to components of the system other than the target,for example, to the capture beads or streptavidin. This can be achievedby subjecting each population of mRNA-protein fusions to at least oneround of fake selection, or “pre-clear”, which consists of incubatingthe library with all the system components except the target molecule.It may also be advantageous to alternate the target capture format. Forexample, each odd-numbered round may employ biotinylated target andstreptavidin beads, and each even-numbered round may employepoxy-immobilized target.

Yeast Surface Display

Yeast surface display is a powerful genetic method that can be used toselect proteins with increased or decreased affinity for anothermolecule, altered specificity, or conditional binding. The geneticmalleability of yeast, the ease of growth of this microbe, and theability to modify binding conditions in vitro combine to produceunprecedented control over the engineering of affinity and specificity.A library of protein variants are displayed on the surface of yeastcells by genetic fusion with yeast secretory and/or anchor proteins in aform accessible for binding to macromolecules, and selected on the basisof binding characteristics using fluorescence activated cell sorting(FACS). In certain embodiments, yeast-displayed proteins are labeled bydyes that fluoresce at two different frequencies. The first dye isconjugated or non-covalently associated with the target of interest; theintensity fluorescent signal at its frequency serves as a measure of thepropensity of the displayed protein to bind the target. The second dyeis conjugated to an antibody that recognizes an epitope tag that is apart of the displayed protein; the intensity fluorescent signal at itsfrequency serves as a measure of the amount of the protein that isdisplayed on the yeast cell.

Yeast surface display offers advantages over bacteria-based technologies(e.g., phage-display) or mRNA display in that yeast can process proteinsthat require endoplasmatic reticulum (ER)-specific post-translationalprocessing for efficient folding and activity. An additional benefit ofyeast surface display is that the protein to be displayed must passthrough the ER and the rest of cellular secretory machinery for assemblyand transport to the cell surface. The efficience of secretion (and thusof display) of a protein depends of the protein biophysical properties,with unstable or aggregation-prone proteins being preferentially removedfrom the pathway before they reach the cell surface (e.g., reference:Hackel et al., J. Mol. Biol., 401:84-96 (2010)). Therefore, yeastsurface display provides the advantage of selecting and isolatingpolypeptides possessing a variety of desirable characteristics (e.g.,thermal stability, desirable expression levels, properly folded,non-aggregating) in addition any other improvement that is selectablevia surface expression of a displayed polypeptide (e.g., target binding,specificity, selectivity). As a consequence, a FACS-gating strategy thatonly captures the yeast cells with the very highest (e.g., the top 5%,the top 2%, the top 1%, or the top 0.1%) intensity of the fluorescentsignal associated with the display level will lead to the selection ofthe proteins with the highest stability and solubility from the mixture.

Any means to display a protein on the surface of yeast is encompassed bythe present disclosure. The different yeast surface display methods allprovide a means of tightly linking each protein encoded by the library(phenotype) to the yeast cell which carries the plasmid encoding thatprotein (genotype). Any stable linkage between phenotype and genotypewill allow the use of yeast surface display in conjunction with mRNAdisplay. Most yeast display methods described to date use the yeastSaccharomyces cerevisiae, but other yeast species, for example, Pichiapastoris, could also be used. There are three commonly used S.cerevisiae based yeast surface display methods: the Aga2-based, theAgα1-based, and the biotin-based surface display methods.

In Aga2-based yeast surface display, a protein, or a library of proteinvariants, is genetically fused to a yeast protein, agglutinin a 2(Aga2), which is naturally associated with yeast cell wall, and to anepitope tag such as, for example, c-myc or HA. Typically, thearrangement of the fusion protein is Aga2-protein variant-tag, butdifferent arrangements have been described. The gene encoding the fusionmay be controlled by an inducible promoter, such as, for example, thegalactose-inducible promoter, GAL1-10. Upon induction, the gene productis secreted from the cell in complex with agglutinin-a-1 (Aga1), withwhich it forms two disulfide bonds. The Aga1, a natural cell wallprotein, is retained in the yeast cell wall, and thus anchors theAga2-protein variant fusion to the cell wall also. The result is aculture where each yeast cell displays between 10,000 and 100,000 copiesof a single protein variant.

In Agα1-based yeast surface display, a protein, or a library of proteinvariants, is genetically fused to a different natural yeast cell-wallprotein, agglutinin alpha 1 (Agα1), and to an epitope tag such as, forexample, c-myc or HA. The gene encoding the fusion may be controlled byan inducible promoter, such as, the galactose-inducible promoter, GAL7.As in Aga2-based yeast surface display, fusion proteins are expressed inthe presence of galactose, secreted from the yeast cells, and retainedon the surface of the yeast cells.

In certain embodiments, a method that relies on in vivo biotinylation ofthe protein to be displayed, followed by its capture on the yeast cellsurface is used. For example, the protein to be displayed is geneticallyfused to a yeast secretory protein of choice and to a biotin-acceptorpeptide (BAP). An epitope tag, such HA or FLAG®, is also engineeredimmediately downstream from the sequence encoding the protein variant.Common secretory proteins include yeast alpha mating factor prepro 1(WTαMFpp), the invertase leader, synthetic leaders (Clements et al.,Gene, 106:267-271 (1991)), and the engineered alpha mating factor preproαMFpp8 (Rakestraw et al., Biotechnol. Bioeng., 103:1192-1201 (2009)).The gene encoding the fusion may be controlled by an inducible promoter,such as, for example, the galactose-inducible promoter, GAL1-10. Beforeinducing expression of the protein to be displayed, the outside surfaceof the yeast cell is chemically conjugated to avidin. Upon induction,the biotin-acceptor peptide in the fusion protein is biotinylated insidethe cell by a co-expressed biotin ligase. The biotinylated fusionprotein is then secreted from the cell and captured on the cell-surfaceavidin due to the extremely high-affinity interaction between avidin andbiotin.

In certain embodiments, the protein variants to be displayed aregenetically fused to a GPI (Glycosyl-Phosphatidyl-Inositol) anchorprotein, such as the mating type protein agglutinin-a-1 (Aga1),flocculin proteins (e.g., Flo1), as well as Sed1, Cwp1, Cwp2, Tip1andTir1/Srp1. In certain embodiments, the anchor protein is selected fromthe group consisting of a GP1 anchor, a modified GP1 anchor, a majorcell wall protein, CCW14, CIS3, CWP1, PIR1, and PIR3.

In order to identify and capture the yeast cells that display proteinvariants with desired properties, the yeast culture is incubated withthe target of interest, then simultaneously labeled by two sets ofreagents: the first set detects the target, and the second set detectsthe protein variant. The target and protein variant may be detectedusing the engineered capture feature. For example, a biotinylated targetcan be detected with streptavidin conjugated with a fluorescent dye, oran Fc fusion can be detected with an anti-Fc specific antibodyconjugated to a fluorescent dye. Typically the protein variant is alsodetected using a fluorescently labeled antibody specific for the epitopetag fused to the protein variant. When using antibodies as detectionreagents, the fluorescent antibody may bind directly to the target,fusion protein, or tag, or an unlabeled primary antibody may bind to thetarget, protein variant, or tag, and the primary antibody is then boundby a labeled secondary antibody. The detection reagents directed at thetarget and at the protein variant are chosen such that they fluoresce atdifferent wavelengths. As the labeled yeast cells are passed through afluorescence-activated cell sorter (FACS), the intensity of signal ateach of the two wavelengths is measured for each cell. The user thendefines the signal range for each wavelength (“the gate”) for whichyeast cells are captured; the rest of the culture is discarded. Thecaptured cells are re-grown, re-labeled, and re-sorted; typically,between four and eight sorts are required to for the population toconverge on a small number of the most successful variants.

In an alternative embodiment, a first fluorescent protein is engineeredimmediately downstream from the sequence encoding the protein variant(e.g., GFP, YFP, etc.). The target protein may also be engineered to befused to a second protein that fluoresces at a different wavelength thanthe first. FACS may then be used to identify and sort the captured yeastcells without the need for the use of labeled detection reagent.

Once an enriched population of yeast-displayed protein variants showsdetectable binding to target under conditions of desired stringency,single cell clones can be isolated and characterized in several,complementary methods. When the primary goal is to identify clones withthe desired binding affinity and specificity, clones can be isolated bygrowth on an agar plate followed by small-scale growth in deep-wellmicroplates. For example, accurate estimates of the dissociationconstant (Kd) can be determined by labeling the yeast cells with aseries of different concentrations of target followed by analytical flowcytometry. Similarly, the off-rate of a particular variant can bemeasured by labeling the clonal culture with labeled target, incubatingwith excess unlabeled target, and using analytical flow cytometry todetermine the fraction of target still bound to yeast at different timepoints. The identity of the protein variants with desired properties canreadily be determined by PCR amplification of the DNA encoding thoseprotein variants from yeast colonies.

When it is necessary to perform more complex assays such as cell-basedfunctional assays and assays for characterizing biophysical properties,purified proteins are produced in a small scale. In cases like these, itmay be advantageous that the starting yeast surface display DNA librarybe expressed in a shuttle plasmid, i.e., a plasmid that can propagate inboth yeast and bacteria. Preferably, the shuttle plasmid can propagatein Saccharomyces cerevisiae and Escherichia coli. In some embodiments,the shuttle-plasmid is extracted from the yeast (e.g., using theZymoprep yeast Plasmid Miniprep II kit by Zymo Research, Irvine, Calif.)and transferred to E. coli. Individual E. coli clones are sequenced toidentify unique selected clones and their level of enrichment. Theselected clones of interest can be expressed in an expression system ofinterest and purified using methods well know in the art. The purifiedvariant proteins of interest can then be further characterized.

Affinity Maturation

Once a target-binding protein has been selected by mRNA display and/oryeast display, the properties of such a clone can be further optimizedby a process named affinity maturation. In affinity maturation, thesequence of the gene encoding a lead protein is re-diversified to anintermediate degree, still retaining some of the sequencecharacteristics of the parent clone but also introducing additionaldiversity. Error-prone PCR and DNA shuffling are two of the manypossible ways of introducing into the sequence such additionaldiversity. For example, after one or more rounds of selection, PCR canbe used to incorporate mutations in the DNA sequences encoding theprotein variants that bind the target of interest. The process, alsoknown as affinity maturation, is typically repeated for several cyclesin order to obtain the peptides having improved affinity for the targetmaterial. Alternatively, gene shuffling may be employed to swapdifferent binding regions within the protein variants. For examples,gene shuffling can be used in connection with a library of ¹⁰Fn3variants to mix and match BC, DE and/or FG loops from different proteinvariants that have been selected to bind to the same target. In certainembodiments, affinity maturation may be used following at least oneround of mRNA display screening, following at least one round of yeastdisplay screening, or both. In certain embodiments, affinity maturationis used after selecting a population of target binding protein variantsusing mRNA display before transitioning the binding protein variantsinto the yeast display system. In certain embodiments, at the end of theprocess, e.g., after suitable binding proteins have been isolated usingrepeated rounds of mRNA display followed by repeated rounds of yeastdisplay.

Polypeptides Comprising ¹⁰Fn3 Domains

In some embodiments, the target binding protein is a ¹⁰Fn3 domain-basedprotein. Fibronectin based scaffold proteins generally make use of ascaffold derived from a fibronectin type III (Fn3) or Fn3-like domainand function in a manner characteristic of natural or engineeredantibodies (that is, polyclonal, monoclonal, or single-chain antibodies)and, in addition, possess structural advantages. Specifically, thestructure of these antibody mimics has been designed for optimalfolding, stability, and solubility, even under conditions that normallylead to the loss of structure and function in antibodies. An example offibronectin-based scaffold proteins are ADNECTINS™ (Adnexus, a whollyowned subsidiary of Bristol-Myers Squibb). Fibronectin-based scaffoldproteins and ADNECTINS™ may be monovalent or multivalent.

An Fn3 domain is small, monomeric, soluble, and stable. It lacksdisulfide bonds and, therefore, is stable under reducing conditions. Theoverall structure of Fn3 resembles the Ig fold. Fn3 domains comprise, inorder from N-terminus to C-terminus, a beta or beta-like strand, A; aloop, AB; a beta or beta-like strand, B; a loop, BC; a beta or beta-likestrand, C; a loop, CD; a beta or beta-like strand, D; a loop, DE; a betaor beta-like strand, E; a loop, EF; a beta or beta-like strand, F; aloop, FG; and a beta or beta-like strand, G. The seven antiparallelβ-strands are arranged as two beta sheets that form a stable core, whilecreating two “faces” composed of the loops that connect the beta orbeta-like strands. Loops AB, CD, and EF are located at one face andloops BC, DE, and FG are located on the opposing face. Any or all ofloops AB, BC, CD, DE, EF and FG may participate in ligand binding. Thereare at least 15 different modules of Fn3, and while the sequencehomology between the modules is low, they all share a high similarity intertiary structure.

The amino acid sequence of the naturally occurring human tenthfibronectin type III domain, i.e., the tenth module of human Fn3(¹⁰Fn3), is set forth in SEQ ID NO: 1:

(SEQ ID NO: 1) VERSUSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKST ATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRT (the AB, CD and EF loops are underlined, and the BC, FG, and DE loopsare emphasized in bold).

In SEQ ID NO:1, the AB loop corresponds to residues 15-16, the BC loopcorresponds to residues 21-30, the CD loop corresponds to residues39-45, the DE loop corresponds to residues 51-56, the EF loopcorresponds to residues 60-66, and the FG loop corresponds to residues76-87. See e.g., Xu et al., Chemistry & Biology, 9:933-942 (2002). TheBC, DE and FG loops align along one face of the molecule (sometimesreferred to as the “north pole” loops) and the AB, CD and EF loops alignalong the opposite face of the molecule (sometimes referred to as the“south pole” loops). In SEQ ID NO: 1, beta strand A corresponds toresidues 9-14, beta strand B corresponds to residues 17-20, beta strandC corresponds to residues 31-38, beta strand D corresponds to residues46-50, beta strand E corresponds to residues 57-59, beta strand Fcorresponds to residues 67-75, and beta strand G corresponds to residues88-94. The strands are connected to each other through the correspondingloop, e.g., strands A and B are connected via loop AB in the formationof strand A, loop AB, strand B, etc. The first 8 amino acids of SEQ IDNO:1 (italicized above) may be deleted while still retaining bindingactivity of the molecule. Residues involved in forming the hydrophobiccore (the “core amino acid residues”) include the amino acidscorresponding to the following amino acids of SEQ ID NO: 1: L8, V10,A13, L18, 120, W22, Y32, 134, Y36, F48, V50, A57, 159, L62, Y68, 170,V72, A74, 188, 190 and Y92, wherein the core amino acid residues arerepresented by the single letter amino acid code followed by theposition at which they are located within SEQ ID NO: 1. See e.g.,Dickinson et al., J. Mol. Biol., 236:1079-1092 (1994).

¹⁰Fn3 domains are structurally analogous to antibodies, specifically thevariable region of an antibody. While ¹⁰Fn3 domains may be described as“antibody mimics” or “antibody-like proteins”, they do offer a number ofadvantages over conventional antibodies. In particular, they exhibitbetter folding and thermostability properties as compared to antibodies,and they lack disulphide bonds, which are known to impede or preventproper folding under certain conditions.

The BC, DE, and FG loops of ¹⁰Fn3 domains are analogous to thecomplementary determining regions (CDRs) from immunoglobulins.Alteration of the amino acid sequence in these loop regions changes thebinding specificity of ¹⁰Fn3. ¹⁰Fn3 domains with modifications in theAB, CD and EF loops may also be made in order to produce a molecule thatbinds to a desired target. The protein sequences outside of the loopsare analogous to the framework regions from immunoglobulins and play arole in the structural conformation of the ¹⁰Fn3. Alterations in theframework-like regions of ¹⁰Fn3 are permissible to the extent that thestructural conformation is not so altered as to disrupt ligand binding.

As described above, amino acid residues corresponding to residues 21-30,51-56, and 76-87 of SEQ ID NO: 1 define the BC, DE and FG loops,respectively. However, it should be understood that not every residuewithin the loop region needs to be modified in order to achieve a ¹⁰Fn3binder having strong affinity for a desired target. For example, in manycases, only residues corresponding to amino acids 23-30 of the BC loopand 52-55 of the DE loop are modified and result in high affinity ¹⁰Fn3binders. Accordingly, in certain embodiments, the BC loop may be definedby amino acids corresponding to residues 23-30 of SEQ ID NO: 1, and theDE loop may be defined by amino acids corresponding to residues 52-55 ofSEQ ID NO: 1. Additionally, insertions and deletions in the loop regionsmay also be made while still producing high affinity ¹⁰Fn3 binders.

Libraries of ¹⁰Fn3 Domain-Based Proteins

Typically, the positions diversified in ¹⁰Fn3-based libraries comprisebetween one and three of the ¹⁰Fn3 loops that are structurally analogousto antibody complementarity-determining regions (CDRs), i.e., loops BC,DE, and FG, the intention being to generate contiguous surfaces fortarget recognition. Diversity is typically introduced into a libraryusing synthetic oligonucleotides containing a mixture of nucleotides.The simplest and least expensive random oligonucleotides incorporate arandom stretches of nucleotides that can encode any amino acid, such asthe so-called NNK codon, where N is an equal mixture of A, T, G and Cand K is a mixture of T and G. Alternatively, oligonucleotidessynthesized from triphosphoramidite or ligated from double-strandedcodons can be used to encode a biased amino-acid composition, forexample, mimicking side-chain distribution in antibody CDRs. In additionto the loop sequence, loop length can be varied by incorporating intothe library synthetic oligonucleotides of divergent lengths. The DNAlibrary encoding the ¹⁰Fn3 variants may be designed to include apromoter (e.g., T7, SP6, T3) for in vitro transcription and atranslation initiation sequence (e.g., Shine Dalgarno for E coli S30extracts, Kozak sequence for eukaryotic systems) for in vitrotranslation, operably linked to the coding sequence. The construction ofa library of variant ¹⁰Fn3 domains having sequence variations in each ofthe BC, DE and FG loops is described in Xu et al., Chemistry & Biology,9:933-942 (2002). Libraries of molecules (e.g., molecules comprising oneor more ¹⁰Fn3 domains or domains relating thereto) that may be screenedare disclosed, e.g., WO 98/056915, WO 02/081497, WO 2008/031098; U.S.Publication No. 2003/186385; WO 2009/102421, WO 2009/142773, WO2010/060095; U.S. Pat. Nos. 6,818,418, 6,673,901, 7,115,396; WO2010/0273261, WO 2011/103105, WO 2011/130354, WO 2011/140086, WO2011/150133, WO 2010/051274, WO 2010/051310, WO 2009/086116, WO2009/086116, WO 2010/093627, WO 2011/137319, WO 2012/016245, WO98/056915, WO 02/081497, WO 2008/031098; U.S. Publication No.2003/186385; WO 2011/130324, WO 2011/130328, WO 2009/083804, WO2009/133208, WO 2010/093627, WO 2011/051333, WO 2011/051466, and WO2011/092233.

To create a library of mRNA-protein fusions for mRNA display, the RNAportion of an RNA-protein fusion may be chemically synthesized usingstandard techniques of oligonucleotide synthesis. Alternatively, the RNAportion may be generated by in vitro transcription of a DNA template.Various RNA polymerases may be used to generate the RNA strandincluding, for example, the SP6, T3 and E. coli RNA polymerases. In onepreferred approach, T7 polymerase is used to enzymatically generate theRNA strand. In addition, the synthesized RNA may be, in whole or inpart, modified RNA. In one particular example, phosphorothioate RNA maybe produced (for example, by T7 transcription) using modifiedribonucleotides and standard techniques. Such modified RNA provides theadvantage of being nuclease stable.

To generate the RNA-protein fusions, any in vitro or in situ translationsystem may be utilized including, for example, eukaryotic translationsystems from lysates from yeast, ascites, tumor cells (Leibowitz et al.,Meth. Enzymol., 194:536 (1991)), and xenopus oocyte eggs, and in vitrotranslation systems from bacterial systems, such as, those described inZubay (Ann. Rev. Genet., 7:267 (1973)); Chen et al. (Meth. Enzymol.,101:44 (1983)); and Ellman (Meth. Enzymol., 202:301 (1991)). Eukaryoticin vitro translation systems are preferred, including, for example, thewheat germ and reticulocyte lysate systems. In principle, however, anytranslation system which allows formation of an RNA-protein fusion andwhich does not significantly degrade the RNA portion of the fusion maybe used. In addition, to reduce RNA degradation in any of these systems,degradation-blocking antisense oligonucleotides may be included in thetranslation reaction mixture; such oligonucleotides specificallyhybridize to and cover sequences within the RNA portion of the moleculethat trigger degradation (see, for example, Hanes et al., Proc. Natl.Acad. Sci. USA, 94:4937 (1997)).

To transition from the mRNA-display system to the yeast display system,the nucleic acid sequence isolated from the mRNA display screening areamplified using 5′ and 3′ primers that introduce sequence overlaps withthe yeast-display plasmid. These sequences are then electroporated intoyeast concurrently with the yeast display plasmid and the proteinvariant sequences are incorporated into the yeast display plasmids byhomologous recombination.

Expression vectors suitable for use in generating the yeast surfacedisplay library may comprise, in a 5′ to 3′ direction, a eukaryoticpromoter, a signal anchor or secretory domain of a protein, and anucleotide sequence that is a cloning site, which allows insertion of agene or gene fragment. Expression vectors for eukaryotic host cellstypically include (i) eukaryotic DNA elements that control initiation oftranscription, such as a promoter, (ii) eukaryotic DNA elements thatcontrol the processing of transcripts, such as a transcriptiontermination/polyadenylation signal sequence, and (iii) optionally,eukaryotic DNA elements that control replication in the eukaryotic hostcell if the vector is to be independently replicated (e.g.,non-integrating vectors). Suitable promoters for expression in yeastinclude promoters from GAL1 (galactose), PGK (phosphoglycerate kinase),ADH (alcohol dehydrogenase), AOX1 (alcohol oxidase), HIS4 (histidinoldehydrogenase), and the like. To ease construction of such expressionvectors, the vectors may optionally include (iv) prokaryotic DNAelements coding for a bacterial replication origin and an antibioticresistance marker to provide for the growth and selection of theexpression vector when manipulating the vector in the bacterial hostcell. Appropriate eukaryotic expression vectors for use with fungal,yeast, and mammalian cellular hosts are known in the art, and aredescribed in, for example, Powels et al. (Cloning Vectors: A LaboratoryManual, Elsevier, NY (1985)). Yeast cloning vectors that may be adaptedfor use in surface display techniques include YIp-based vectors, such asYIp5, YRp vectors, such as YRp17, YEp vectors such as YEp13 and YCpvectors, such as YCp19. Yeast species that are suitable for surfacedisplay techniques include Saccharomyces cerevisiae, Pichia pastoris,and Pichia methanolica.

EXAMPLES

The invention now being generally described will be more readilyunderstood by reference to the following examples which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention inany way.

Example 1 Selection and Characterization of PCSK9-Binding ¹⁰Fn3 DomainProteins Having Favorable Biophysical Properties

To evaluate the benefit of combining mRNA display and yeast surfacedisplay, PCSK9-binding proteins were selected from a 10¹²-memberedlibrary of variants of the ¹⁰Fn3 domain. Two different selectionstrategies were applied to the library in parallel:

1. mRNA display only (“R”). In this approach, the population wassubmitted to five consecutive rounds of mRNA display, all of which wereperformed at the PCSK9 target concentration of 100 nM. Nucleic-acidmixtures obtained after round 5 were subcloned into the E. coli vectorpET9d. Approximately 2,100 clones were sequenced, and approximately1,300 unique clones were expressed in E. coli and screened for PCSK9binding. For this study, 89 of the 934 PCSK9-binding ¹⁰Fn3-basedproteins were evaluated for state of aggregation and thermostability.

2. mRNA display followed by yeast surface display (“R+Y”). In thisapproach, the population that had undergone the first three rounds ofmRNA-display selection described under (1) was transferred into a yeastsurface display system, and subjected to five rounds of yeast surfacedisplay, also at the target concentration of 100 nM of PCSK9. Thesorting criteria in yeast surface display are illustrated in FIG. 1,which shows the gates applied to labeled yeast surface populations inround 1. After each round of yeast surface display, plasmid DNA wasextracted from the enriched culture; the ¹⁰Fn3 domain-encoding sequencewas subcloned from the yeast-display plasmid into the E. coli vectorpET9d, and 96 clones per round were sequenced. All clones enriched tomore than 1% of the population after round 4 or round 5 were expressedin E. coli and screened for PCSK9 binding, state of aggregation, andthermostability. In addition, 20% of the clones that were detected bysequencing, at any frequency, in rounds 2 to 5 were also screened forthe same properties.

When comparing approaches R and R+Y, the main focus was on twobiophysical properties of the enriched clones:

1. State of association as determined by analytical size-exclusionchromatography. The size-exclusion chromatography trace of each¹⁰Fn3-based protein was scored with a single number (“SEC score”)according to the following criteria:

-   -   a. SEC score 1: All monomeric,    -   b. SEC score 2: Mostly monomeric, detectable amount of oligomer,    -   c. SEC score 3: Mostly oligomeric, detectable amount of monomer,        and    -   d. SEC score 4: Mostly aggregated. 2. Melting temperature        (T_(m)) as determined by differential scanning calorimetry. Only        ¹⁰Fn3 domain proteins with SEC scores of 1, 2, or 3 were        subjected to differential scanning calorimetry.

The comparison of SEC scores between the two populations (FIG. 2) showsthat the combined (R+Y) approach led to an approximately two-folddecrease relative to mRNA display only (R) in the fraction of highlyaggregated ¹⁰Fn3 domain proteins (SEC score 4), to a four-fold increasein fraction of mostly oligomeric ¹⁰Fn3 domain proteins with somemonomer, and to a seven-fold increase in fraction of mostly monomeric¹⁰Fn3 domain proteins. Given that the monomeric state is preferred inprotein therapeutics, this data demonstrates that the addition of yeastsurface display to mRNA display can increase the efficiency of drugdiscovery.

The difference in T_(m) values between the two populations (FIG. 3) issmaller and harder to interpret due to a small number of clones tested.Nevertheless, the identification of a new ¹⁰Fn3 domain protein with amelting temperature of 87° C. using the (R+Y) approach also suggeststhat the use of a combination of mRNA display and yeast surface displayis preferable to the use of mRNA display alone.

Example 2 Selection and Characterization of ¹⁰Fn3 Domain ProteinsBinding a Protein Target and Having Favorable Biophysical Properties

To evaluate the benefit of combining mRNA display and yeast surfacedisplay, proteins binding to a target protein, which is different fromthat in Example 1, were selected from a 10¹²-membered library ofvariants of the ¹⁰Fn3 domain. Two different selection strategies wereapplied to the library in parallel:

1. mRNA display only (“R”). In this approach, the population wassubmitted to five consecutive rounds of mRNA display, all of which wereperformed at the target concentration of 100 nM. Nucleic-acid mixturesobtained after rounds 2 and 3 were subcloned into the E. coli vectorpET9d and evaluated for state of aggregation and thermostability.

2. mRNA display followed by yeast surface display (“R+Y”). In thisapproach, the population that had undergone the first two and the firstthree rounds of mRNA-display selection described under (1) wastransferred into a yeast surface display system, and subjected to fiverounds of yeast surface display. The first round of sorting wasperformed for display level only, and rounds 2 to 5 were performed atthe target concentration of 100 nM. The sorting criteria in yeastsurface display are illustrated in FIG. 4, which shows the gates appliedto labeled yeast surface populations in round 3. After rounds 3, 4, and5 of yeast surface display, plasmid DNA was extracted from the enrichedculture; the ¹⁰Fn3 domain-encoding sequence was subcloned from theyeast-display plasmid into the E. coli vector pET9d, sequenced,expressed in E. coli and screened for target binding, state ofaggregation, and thermostability.

When comparing approaches R and R+Y, the main focus was on twobiophysical properties of the enriched clones:

1. State of association as determined by analytical size-exclusionchromatography. The size-exclusion chromatography trace of each¹⁰Fn3-based protein was scored with a single number (“SEC score”)according to the following criteria:

-   -   a. SEC score 1: All monomeric,    -   b. SEC score 2: Mostly monomeric, detectable amount of oligomer,    -   c. SEC score 3: Mostly oligomeric, detectable amount of monomer,        and    -   d. SEC score 4: Mostly aggregated. 2. Melting temperature (half        point of thermal transition;T_(H)) as determined by        fluorescence-detected thermal denaturation, where the        temperature at the half-point of the denaturing thermal        transition was recorded.

The comparison of SEC scores between the two populations (FIG. 5) showsthat the combined (R+Y) approach led to a 2½-fold decrease relative tomRNA display only (R) in the fraction of highly aggregated ¹⁰Fn3 domainproteins (SEC score 4); an 8 fold increase relative to the mRNA displayonly in the fraction of all monomeric protein (SEC score 1); an 8 foldincrease relative to the mRNA display only in the fraction of mostlymonomeric protein (SEC score 2); and a 2 fold increase relative to themRNA display only in the fraction of mostly oligomeric protein (SECscore 3). Given that the monomeric state is preferred in proteintherapeutics, this data again demonstrates that the addition of yeastsurface display to mRNA display can increase the efficiency of drugdiscovery.

In this case, the difference in T_(H) values between the two populations(FIG. 6) is as dramatic as the difference between their propensity toaggregate. Particular attention is drawn to the region in the boxedarea, which highlights the proteins with both a high melting temperature(higher or equal to 60° C.) and a completely or mostly monomeric state(SEC score 1 or 2). Proteins in this region represent the most promisingdrug candidates; the addition of yeast surface display to the selectionprotocol increased their number 30 fold.

INCORPORATION BY REFERENCE

The entire disclosure of each document cited (including patents, patentapplications, journal articles, abstracts, laboratory manuals, books,GENBANK® Accession numbers, SWISS-PROT® Accession numbers, or otherdisclosures) in the Background, Detailed Description, Brief Descriptionof Drawings, and Examples is hereby incorporated herein by reference intheir entirety.

The present invention is not to be limited in scope by the embodimentsdisclosed herein, which are intended as single illustrations ofindividual aspects of the invention, and any that are functionallyequivalent are within the scope of the invention. Various modificationsto the models and methods of the invention, in addition to thosedescribed herein, will become apparent to those skilled in the art fromthe foregoing description and teachings, and are similarly intended tofall within the scope of the invention. Such modifications or otherembodiments can be practiced without departing from the true scope andspirit of the invention.

I claim:
 1. A method for selecting at least one ¹⁰Fn3 target-bindingprotein with favorable biophysical properties, comprising a first phasecomprising selecting a first population of ¹⁰Fn3 variants binding atarget protein (“target-binding protein variants”) from a librarycomprising at least 10¹² ¹⁰Fn3 variants, wherein the first phasecomprises performing a number of rounds of selection of the librarysufficient to obtain from 10⁴ to 10⁸ ¹⁰Fn3 target-binding proteinvariants, wherein the library of the first phase is a ¹⁰Fn3 library ofmRNA-protein fusions; and a second phase comprising selecting a secondpopulation of ¹⁰Fn3 target-binding protein variants from at least aportion of the first population of ¹⁰Fn3 target-binding protein variantsusing yeast surface display, wherein the second population of ¹⁰Fn3target-binding protein variants comprises ¹⁰Fn3 target-binding proteinwith favorable biophysical properties, wherein the favorable biophysicalproperties are one or more of solubility, thermostability, highexpression levels, low levels of aggregation, proper folding, or bindingspecificity, wherein the second phase comprises performing a number ofrounds of selection on at least a portion of the 10⁴ to 10⁸ ¹⁰Fn3target-binding protein variants selected from the first phase sufficientto obtain at most 10³ ¹⁰Fn3 target-binding protein variants.
 2. Themethod of claim 1, wherein the library comprises at least 10¹³ proteinvariants.
 3. The method of claim 1, wherein the first phase comprises atleast one round of selection, wherein a round of selection comprisescontacting the target protein with the library of protein variants,removing any protein variants that do not bind to the target with adesired affinity, and isolating the remaining protein variants.
 4. Themethod of claim 3, wherein the first phase comprises at least 2consecutive rounds of selection.
 5. The method of claim 4, wherein thefirst phase comprises at least 3 to 4 consecutive rounds of selection.6. The method of claim 1, wherein the mRNA-protein fusions arecovalently conjugated via a puromycin linker.
 7. The method of claim 3,wherein the second phase comprises at least one round of selection,wherein a round of selection comprises contacting the target proteinwith the first population of ¹⁰Fn3 target-binding protein variantsdisplayed on yeast cells, removing any yeast cells displaying proteinvariants that do not bind to the target with a desired affinity, tothereby select the second population of ¹⁰Fn3 target-binding proteinvariants.
 8. The method of claim 7, wherein the second phase comprisesat least 2 consecutive rounds of selection.
 9. The method of claim 8,wherein the second phase comprises at least 3 to 6 consecutive rounds ofselection.
 10. The method of claim 7, wherein the second phase comprisesperforming a number of rounds of selection sufficient to obtain at most10² ¹⁰Fn3 target-binding protein variants.
 11. The method of claim 7,wherein the second phase comprises at least one round of selectionwherein at most the top 10% of the yeast cells that express the highestlevel of ¹⁰Fn3 target-binding protein variants are selected.
 12. Themethod of claim 11, wherein each of the rounds of selection of yeastsurface display comprise selecting at most the top 10% of the yeastcells that express the highest level of ¹⁰Fn3 target-binding proteinvariants.